Article pubs.acs.org/ac
Ultrasensitive Detection of Transcription Factors Using Transcription-Mediated Isothermally Exponential AmplificationInduced Chemiluminescence Fei Ma,† Yong Yang,† and Chun-yang Zhang* Single-Molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Guangdong 518055, China S Supporting Information *
ABSTRACT: Transcription factors (TFs) are important cellular components that modulate gene expression, and the malregulation of transcription will lead to a variety of diseases such as cancer and developmental syndromes. However, the conventional methods for transcription factor assay are generally cumbersome and costly with low sensitivity. Here, we develop a label-free strategy for ultrasensitive detection of transcription factors using a cascade signal amplification of RNA transcription, dual isothermally exponential amplification reaction (EXPAR), and G-quadruplex DNAzyme-driven chemiluminescence. Briefly, the specific binding of TF with the detecting probe prevents the cleavage of the detecting probe by exonuclease and subsequently facilitates the conversion of TF signal to abundant RNA triggers in the presence of T7 RNA polymerase. The obtained RNA triggers can initiate the strand displacement amplification to yield abundant DNAzymes and DNA triggers, and the released DNA triggers can further initiate the next rounds of EXPAR reaction. The synergistic operation of dual EXPAR reaction can produce large amounts of DNAzymes, which subsequently catalyze the oxidation of luminol by H2O2 to yield an enhanced chemiluminescence signal with the assistance of cofactor hemin. Conversely, in the absence of target TF, the naked detecting probes will be completely digested by exonucleases, leading to neither the transcription-mediated EXPAR nor the DNAzyme-driven chemiluminescence signal. This method has a low detection limit of as low as 6.03 × 10−15 M and a broad dynamic range from 10 fM to 1 nM and can even measure the NF-κB p50 of crude cell nuclear extracts. Moreover, this method can be used to measure a variety of DNA-binding proteins by simply substituting the target-specific binding sequence in the detecting probes.
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INTRODUCTION Transcription factors (TFs) are a class of DNA-binding proteins that modulate the flow of genetic information from DNA to RNA.1 TFs can recognize small degenerate DNA sequences (6−12 bp) within the gene regulatory region and generally function in combinatorial manners to regulate the transcription of target genes. 2 Regular transcription is indispensable for normal cellular function, accurate embryonic development,3,4 the maintenance of cell type- and tissuespecific gene expression,5−7 and the response to cellular signaling,8,9 whereas the transcription misregulation10 and the mutations in transcription factors11 might lead to a variety of diseases such as cancer,12 diabetes,13 congenital heart disease,14 autoimmune diseases,15,16 and developmental syndromes.2 Owing to the pivotal role of TFs in gene expression as well as its close relationship with human diseases, the accurate measurement of transcription factors is of great importance to both biological research and medical diagnosis. The conventional methods for transcription factor measurement are electrophoretic mobility shift assay (EMSA)17 and footprinting analysis.18 Despite their widespread applications, © 2014 American Chemical Society
these methods are generally tedious and cumbersome with low throughput.19,20 The enzyme-linked immunosorbent assay (ELISA) is relatively sensitive,21 but it requires the specific antibody against the target. To overcome these problems, a homogeneous molecule beacon assay has been developed.19 However, its application is limited due to its reliance on the rational design of a covalent break within the TF binding site.22,23 To improve the detection sensitivity, some DNA amplification-based approaches have been developed, including the real-time polymerase chain reaction (PCR) assay,24 the isothermally exponential amplification-based colorimetric assay,20 the target-converted helicase-dependent amplification assay,25 and the near-infrared fluorescent solid phase-based rolling circle amplification.26 These methods can amplify the TFs signal effectively. In view of the large numbers of transcription factors being identified27 and their nonuniform expression in different tissues,5,28,29 a versatile and sensitive Received: March 27, 2014 Accepted: May 27, 2014 Published: May 27, 2014 6006
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England Biolabs (Beverly, MA). The T7 RiboMAX Express Large Scale RNA Production System was obtained from Promega (Madison, WI,). Both SYBR Gold and SYBR Green I was purchased from Xiamen Bio-Vision Biotechnology (Xiamen, China). Protein−DNA Interaction and Exo III Digestion. The NF-κB p50 protein at various concentrations were mixed with 1 μL of 10 μM probes in 10 μL of binding buffer (10 mM TrisHCl, pH 7.5, 100 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mg/mL yeast tRNA, 10% glycerol, 0.25 mM DTT) and incubated at 37 °C for 20 min. Then 0.5 μL of Exo III (100 U/ μL) and 1 μL of 10 × NEB buffer 1 were added to the mixture and incubated for another 20 min at 37 °C to remove the free probes, followed by heating at 80 °C for 10 min to inactive Exo III. DNA Transcription. Before in vitro transcription, 1 μL of Exo I (20 U/μL) was added to the Exo III-treated solution and incubated at 37 °C for 20 min to remove the overhangs of the probes, followed by termination at 80 °C for 10 min. The DNA transcription was carried out in 20 μL of solution containing 2 μL of the Exo I-digested products at 37 °C for 60 min in T7 RiboMAX Express Large Scale RNA Production System (Promega, USA). EXPAR. The EXPAR reaction mixture was composed of part I and part II. Part I contained 2 μL of RNA transcription products, 2 μL of EXPAR template (1 μM), 0.5 μL of deoxynucleotide triphosphates (10 mM), and 2 μL of 10× NEB buffer 2. Part II contained 0.8 μL of Nt.BstNBI nicking endonuclease (10 U/μL) and 0.5 μL of Vent (exo−) DNA polymerase (2 U/μL) and 12.2 μL of H2O. The reaction was carried out in 20 μL of reaction solution. Before mixing with part II, part I was heated at 95 °C for 3 min. After the mixing of part I with part II, the reaction was performed at 55 °C for 50 min. The obtained products were kept at 4 °C for further use. Real-Time Fluorescence Measurement and Chemiluminescence Assay. The real-time fluorescence measurement was performed in the Roche Light Cycler Nano (Switzerland), and the fluorescence signal was recorded at a time interval of 30 s with SYBR Green I as the fluorescent indicator. For chemiluminescence assay, 20 μL of luminol solution (2.5 mM) and 40 μL of hemin solution (250 nM) were mixed with 20 μL of EXPAR products, 20 μL of distilled H2O, and 75 μL of reaction buffer (40 mM HEPES, pH 8.0, 300 mM NaCl, 20 mM KCl), followed by incubation at room temperature for 30 min to allow the obtained DNAzymes to fold into active quadruplex structure. Then 25 μL of H2O2 (80 mM) was added to the above solution, followed by the measurement of chemiluminescence signals using the GloMax 96 Microplate Luminometer (Promega, Madison, WI) with an interval of 1.5 s. Preparation of HeLa Cell Nuclear Extracts. HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, USA) with 10% fetal bovine serum (FBS; Invitrogen, USA) and 50 U/mL of penicillin plus 50 μg/ mL of streptomycin at 37 °C with 5% CO2. For the H2O2treatment experiment, the HeLa cells were treated with 500 μM H2O2 for 30 min before harvesting. The nuclear extracts were prepared using the nuclear extract kit (ActiveMotif, Carlsbad, CA), and the concentration of proteins was measured using Bradford-based assay. The activity of endogenous NF-κB p50 was determined by EMSA as well. Briefly, 2 μL of detecting probe (10 μM) and the nuclear extracts were incubated in 20 μL of binding buffer (10 mM Tris-HCl, pH 7.5, 100 mM KCl,
method that can measure transcription factors with a large dynamic range is highly desired. Recently, the functional G-quadruplex DNAzymes are widely used to measure a variety of targets owing to their high sensitivity, easy synthesis, and the capability to minimize nonspecific adsorption.30,31 Upon complexation with hemin,32 the G-quadruplex DNAzymes exhibit an enhanced horseradish peroxidase (HRP)-mimicking activity for 2,2′-azino-bis(3ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS) and luminol, generating a colored product32 and a chemiluminescence signal,33 respectively. The DNAzymes have been used to measure DNAs,30,33 metal ions,34,35 proteins,36 and the monitoring of telomerase activity.37 In this research, we demonstrate a label-free method for ultrasensitive measurement of transcription factors by integrating transcription-mediated dual isothermally exponential amplification reaction and Gquadruplex DNAzyme-driven chemiluminescence. Due to the cascade signal amplification of RNA polymerase-mediated transcription,38 the dual isothermally exponential amplification reaction,20,39 and the G-quadruplex/hemin DNAzyme-catalyzed chemiluminescence,33 the proposed method can detect NF-κB p50 with an extremely low detection limit of 6.03 × 10−15 M and a large dynamic range over 5 orders of magnitude, and it can be further applied to measure endogenous NF-κB p50 of crude cell nuclear extracts.
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EXPERIMENTAL SECTION Materials. The oligonucleotides (Table 1), RNase inhibitor, DEPC-treated water, and four mixed nucleotides (dNTPs) Table 1. Sequences of Synthesized Oligonucleotidesa note p50-Sb p50-ASc m-p50-S m-p50-AS EXPAR template synthesized DNAzyme
sequence (5′−3′) AGA TGG GAC TTT CCT TGC TAA TAC GAC TCA CTA TAG GGT CTC AGT CGT AGT GAG TCT GTT CTT GAT AAC AGA CTC ACT ACG ACT GAG ACC CTA TAG TGA GTC GTA TTA GCA AGG AAA GTC CCA TCT AGA TCT CAC TTT CCT TGC TAA TAC GAC TCA CTA TAG GGT CTC AGT CGT AGT GAG TCT GTT CTT GAT AAC AGA GTC ACT ACG ACT GAG ACC CTA TAG TGA GTC GTA TTA GCA AGG AAA GTG AGA TCT CCC AAC CCG CCC TAC CCA ACA GAC TCA CTA CGA CTG AGA CCC AAC AGA CTC ACT ACG ACT GAG ACC C−P GGG TAG GGC GGG TTG GG
a The boldface bases represent the binding region of NF-κB p50, and the underlined bold bases indicate the mutant bases. The italic bold bases indicate the T7 promote sequence. The underlined bases within the EXPAR template are the specific recognition sites of Nt.BstNBI nicking endonuclease. bThe letter S refers to the sense strand of the probe. cAS refers to the antisense strand of the probe.
were obtained from Takara Biotechnology Co. Ltd. (Dalian, China). To prepare the double-stranded detecting probe, two relevant single-stranded oligonucleotides were mixed at an equal molar ratio in the hybridization buffer (50 mM Tris− HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA). After heating at 95 °C for 5 min, the mixture was cooled to room temperature. The NF-κB p50 protein and c-Jun protein were purchased from Cayman Chemical (Ann Arbor, MI) and ProteinOne (Bethesda, MD), respectively. The exonuclease III (Exo III), exonuclease I (Exo I), Vent (exo−) DNA polymerase, and the Nt.BstNBI nicking endonuclease were purchased from New 6007
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endonuclease Nt.BstNBI,39 generating abundant DNA triggers and DNAzymes. Meanwhile, the released DNA triggers can further react with the EXPAR templates to initiate the next EXPAR. This dual EXPAR reaction will release large amounts of free DNAzymes to catalyze the H2O2-mediated oxidation of luminol, yielding an enhanced chemiluminescence signal in the presence of hemin. Conversely, in the absence of the TF, the probes will be degraded by exonuclease III and exonuclease I, resulting in neither the transcription-mediated EXPAR nor the DNAzyme-driven chemiluminescence signal. As a proof of concept, we demonstrated the quantitative measurement of transcription factor NF-κB p50. Validation of the Assay. The primary aspect of this assay is the conversion of the TF signal into RNA molecules. To determine whether the binding between TF and the probe can prevent the probe from Exo degradation and subsequently initiate the transcription reaction, we used a nondenaturing gel electrophoresis to monitor the transcription product generated by T7 RNA polymerase.38 As shown in Figure 1A, two distinct
2 mM MgCl2, 0.1 mM EDTA, 0.1 mg/mL yeast tRNA, 10% glycerol, and 0.25 mM DTT) at room temperature for 30 min. After labeling with SYBR Gold (1 μg/mL), the mixture was resolved on a prerun 8% nondenaturing polyacrylamide gel at 110 V in Tris-borate-EDTA (TBE) buffer (9 mM Tris-HCl, pH 7.9, 9 mM boric acid, and 0.2 mM EDTA). The gel was scanned by the Kodak 4000 MM imaging station (Kodak, Japan). Gel Electrophoresis. The products of two-stage isothermal amplification were resolved on a 12% nondenaturing polyacrylamide gel and run at room temperature in TBE buffer (9 mM Tris-HCl, pH 7.9, 9 mM boric acid, 0.2 mM EDTA) with a 110 V constant voltage for 50 min. The gel was stained by SYBR Gold and scanned with the Kodak Image Station 4000 MM.
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RESULTS AND DISCUSSION Principle of Transcription Factor Assay. The proposed method for transcription factor assay involves the TF-binding probe, exonuclease III/I (Exo III/I), T7 RNA polymerase, EXPAR template, and HRP-mimicking DNAzyme of PW17 (Scheme 1). The TF-binding probe is a double-stranded DNA
Scheme 1. Schematic Representation of Transcription Factor Assaya
a
This method combines the transcription-mediated dual exponential amplification reaction with the DNAzyme-catalyzed chemiluminescence.
Figure 1. (A) Analysis of the transcription products by nondenaturing PAGE. Lanes M, 1, and 2 show the DNA ladder marker, the products without the addition of NF-κB p50, and the products with the addition of 10 nM NF-κB p50, respectively. (B) Analysis of the EXPAR products by nondenaturing PAGE. Lanes M, 1, and 2 show the DNA ladder marker, the products without the addition of NF-κB p50, and the products with the addition of 1 nM NF-κB p50, respectively. (C) Time-dependent fluorescence signals obtained from dual EXPAR reaction in response to 1 nM NF-κB p50 (red line) and the control group without NF-κB p50 (black line). (D) Quantification of DNAzyme-catalyzed chemiluminiscence signal in response to 1 nM NF-κB p50 (red column) and the control group without NF-κB p50 (black column). Error bars are standard derivation obtained from three independent experiments.
in which the sense strand bears a specific TF recognition element and the T7 promoter sequence at the 5′ terminus and exonuclease III-resistant 3′-protruding termini (6 bases) at the 3′ terminus (p50-S in Table 1). In the presence of TFs, the binding of the probes with TFs prevents the probes from degradation by exonuclease III and exonuclease I (exonuclease III can remove the mononucleotides from 3′-hydroxyl termini of a duplex DNA,40 while exonuclease I can degrade the singlestranded DNA41,42). The probes with blunt ends can be efficiently transcribed by T7 RNA polymerase to yield abundant RNA,38 which is complementary to region I of the EXPAR template, and subsequently trigger the EXPAR reaction. The EXPAR template (Table 1) is a single-stranded DNA that contains three regions separated by two nicking endonuclease cutting sites, where regions I and II have an identical sequence and region III is complementary to the sequence of PW17 DNAzyme (see synthesized DNAzyme in Table 1). Upon hybridization with the EXPAR template, the transcribed RNA initiates the RNA-induced EXPAR with the assistance of Vent (exo−) DNA polymerase and nicking
bands of 25 nt and 60 bp, which represent the transcription product and the blunt probe, respectively, are observed with the addition of NF-κB p50 (Figure 1A, lane 2). However, neither the templates nor the RNA amplicons can be observed in the control group without the addition of NF-κB p50 (Figure 1A, lane 1). To verify whether the transcribed RNA amplicons can trigger the subsequent EXPAR reaction,39 we further measured the products of the EXPAR reaction with a nondenaturing PAGE. As shown in Figure 1B, a distinct band of G-quadruplex 6008
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helicase-dependent amplification method (0.93 pM).25 The improved sensitivity of the current assay might be ascribed to two factors: (1) the cascade signal amplification induced by DNA transcription,38 EXPAR reaction, and G-quadruplex DNAzyme-driven chemiluminescence33 and (2) abundant DNAzymes produced by the transcription-mediated dual EXPAR amplification.20,39 Specificity of the Assay. The specificity of current assay depends primarily on the specific binding of TF with the probes. To investigate the detection specificity, we measured the chemiluminescence intensity induced by a nonspecific binding probe, in which the p50 binding sequence of GGG AC TTT was replaced by CTC AC TTT (Table 1) and two distinct proteins including an irrelevant protein BSA20 and an oncogenic protein c-Jun45 under identical conditions (Figure 3). An extremely high chemiluminescence intensity is obtained
DNAzymes (17 nt) is observed with the addition of NF-κB p50 (Figure 1B, lane 2), while no distinguishable band of Gquadruplex DNAzymes is observed in the control group without the addition of NF-κB p50 (Figure 1B, lane 1), indicating that the transcribed RNA can initiate the EXPAR reaction effectively and specifically. These results are also confirmed by the real-time fluorescence assay (Figure 1C). The addition of NF-κB p50 induces an increasing fluorescence signal in the initial 50 min, followed by reaching a plateau (Figure 1C, red line). In contrast, no enhanced fluorescence is detected in the control group without the addition of NF-κB p50 (Figure 1C, black line). Furthermore, we examined whether the obtained DNAzymes can form an active Gquadruplex structure with the cofactor hemin to catalyze the luminol-H2O2 reaction (Figure 1D). A strong chemiluminescence signal is detected with the addition of NF-κB p50 (Figure 1D, red column), while no distinct chemiluminescence signal is detected in the control group without NF-κB p50 (Figure 1D, black column). Improved Sensitivity of the Assay. We further measured NF-κB p50 at different concentrations using the proposed chemiluminiscence assay. Under optimal conditions (Figure S1 in Supporting Information), the chemiluminescence (CL) signal increases with the concentration of NF-κB p50, and the CL intensity shows logarithmical correlation with the concentration of NF-κB p50 in the range from 10 fM to 1 nM (Figure 2). The correlation equation is I = 6.97 E6 + 4.84E5
Figure 3. Chemiluminescence signals induced by 1 nM NF-κB p50 and 1 μM nonspecific probes (control, black column), 1 nM BSA and 1 μM NF-κB p50-specific probes (BSA, blue column), 1 nM c-Jun protein and 1 μM NF-κB p50-specific probes (c-Jun, green column), and 1 nM NF-κB p50 and 1 μM NF-κB p50-specific probes (p50, red column), respectively. Error bars are standard derivation obtained from three independent experiments.
only in response to the coexistence of NF-κB p50 and NF-κB p50-specific probes (Figure 3, red column), whereas no significant chemiluminescence signal is detected in response to either a nonspecific probe (Figure 3, black column) or nonspecific proteins such as BSA (Figure 3, blue column) and c-Jun protein (Figure 3, green column), confirming the excellent specificity of the current assay toward NF-κB p50. Measurement of Endogenous NF-κB p50. The nuclear extracts contain various DNA-binding proteins that may interfere with the NF-κB p50 assay.24 We further challenged our method by measuring the endogenous NF-κB p50 in crude HeLa cell nuclear extracts, and we used the conventional EMSA assay to validate the activity of NF-κB p50 as well (Figure 4). Without the addition of NF-κB p50-specific probes, no distinguishable band is observed in crude nuclear extracts (Figure 4A, lane 1). While with the addition of NF-κB p50specific probes, two distinct bands of NF-κB p50-probe complexes and free probes are observed (Figure 4A, lane 2), and a remarkable chemiluminescence signal is obtained correspondingly (Figure 4B, black column), suggesting the feasibility of the current method for cellular NF-κB p50 assay. Interestingly, the treatment of HeLa cells with H2O2, which can induce oxidative stress in the cells, generates a stronger band of NF-κB p50-probe complex (Figure 4A, lane 3) and correspondingly a higher chemiluminescence signal (Figure
Figure 2. Chemiluminescence (CL) intensity shows a linear relationship with the logarithm of the concentrations of NF-κB p50. Error bars are standard derivation obtained from three independent experiments.
log10 C (R2 = 0.9945), where I and C represents the CL intensity and the concentration of NF-κB p50, respectively. The calculated detection limit is 6.03 × 10−15 M, which is 10-fold lower than that obtained by real-time fluorescence assay (Figure S2 in Supporting Information). This enhanced sensitivity may be ascribed to the HRP-mimicing property of the DNAzyme−hemin complex which can catalyze the H2O2luminol reaction to enlarge the signal.33 It is worth noting that the sensitivity of current assay has improved by 6 orders of magnitude in comparison with those of molecular beacon-based FRET assay (20 nM),19 the label-free luminescence switch-on method (30 nM),43 and the gold nanoparticle-based colorimetric assay (10 nM).44 In addition, the sensitivity of the current assay has improved by 2 orders of magnitude in comparison with the isothermally exponential amplificationbased colorimetric assay (3.8 pM)20 and the target-converted 6009
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ACKNOWLEDGMENTS We gratefully acknowledge the financial supports from the Natural Science Foundation of China (Grants 21325523), the Award for the Hundred Talent Program of the Chinese Academy of Sciences, and the Fund for Shenzhen Engineering Laboratory of Single-molecule Detection and Instrument Development (Grant No. (2012)433).
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Figure 4. (A) EMSA analysis of the activity of endogenous NF-κB p50 in HeLa cell nuclear extracts. Lane 1 is 10 μg of nuclear extracts without the detecting probes, lane 2 is 10 μg of nuclear extracts with 1 μM NF-κB p50-specific probes, and lane 3 is 10 μg of H2O2-treated nuclear extracts with 1 μM NF-κB p50-specific probes. (B) Measurement of the chemiluminescence signals in normal HeLa cell nuclear extracts (black column), and H2O2-treated HeLa cell nuclear extracts (red column). Error bars are standard derivation obtained from three independent experiments.
4B, red column) than the untreated cells (Figure 4A, lane 2 and Figure 4B, black column), suggesting the enhanced activity of endogenous NF-κB p50 induced by oxidative stress46 as well as the feasibility of the current method for the measurement of NF-κB p50 in real samples.
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CONCLUSIONS In conclusion, we have demonstrated a label-free and ultrasensitive method for transcription factor assay using transcription-mediated dual EXPAR and G-quadruplex DNAzyme-driven chemiluminescence. The specific binding of TF with the detecting probes can not only ensure the detection specificity, but also trigger the subsequent cascade signal amplification of RNA transcription, dual EXPAR, and the DNAzyme-catalyzed chemiluminescence. The detection limit of current assay can reach as low as 6.03 × 10−15 M, which is more sensitive than those of previously reported methods.19,20,25,43,44 Notably, the detecting probe used in this research does not require any modification, and the entire reaction can be carried out in an isothermal manner without the involvement of highprecision thermal cycler, making current assay extremely costeffective. More importantly, this method can be used to measure a variety of DNA-binding proteins by simply substituting the target-specific DNA-binding sequence in the detecting probes.
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ASSOCIATED CONTENT
S Supporting Information *
Optimization of experimental conditions and the real-time fluorescence measurement. This material is available free of charge via the Internet at http://pubs.acs.org.
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REFERENCES
AUTHOR INFORMATION
Corresponding Author
*Phone: +86-755-8639-2211. Fax: +86-755-8639-2299. E-mail: [email protected]. Author Contributions †
These authors contributed equally.
Notes
The authors declare no competing financial interest. 6010
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Ultrasensitive Detection of Transcription Factors Using Transcription-Mediated Isothermally Exponential AmplificationInduced Chemiluminescence Fei Ma,† Yong Yang,† and Chun-yang Zhang* Single-Molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Guangdong 518055, China S Supporting Information *
ABSTRACT: Transcription factors (TFs) are important cellular components that modulate gene expression, and the malregulation of transcription will lead to a variety of diseases such as cancer and developmental syndromes. However, the conventional methods for transcription factor assay are generally cumbersome and costly with low sensitivity. Here, we develop a label-free strategy for ultrasensitive detection of transcription factors using a cascade signal amplification of RNA transcription, dual isothermally exponential amplification reaction (EXPAR), and G-quadruplex DNAzyme-driven chemiluminescence. Briefly, the specific binding of TF with the detecting probe prevents the cleavage of the detecting probe by exonuclease and subsequently facilitates the conversion of TF signal to abundant RNA triggers in the presence of T7 RNA polymerase. The obtained RNA triggers can initiate the strand displacement amplification to yield abundant DNAzymes and DNA triggers, and the released DNA triggers can further initiate the next rounds of EXPAR reaction. The synergistic operation of dual EXPAR reaction can produce large amounts of DNAzymes, which subsequently catalyze the oxidation of luminol by H2O2 to yield an enhanced chemiluminescence signal with the assistance of cofactor hemin. Conversely, in the absence of target TF, the naked detecting probes will be completely digested by exonucleases, leading to neither the transcription-mediated EXPAR nor the DNAzyme-driven chemiluminescence signal. This method has a low detection limit of as low as 6.03 × 10−15 M and a broad dynamic range from 10 fM to 1 nM and can even measure the NF-κB p50 of crude cell nuclear extracts. Moreover, this method can be used to measure a variety of DNA-binding proteins by simply substituting the target-specific binding sequence in the detecting probes.
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INTRODUCTION Transcription factors (TFs) are a class of DNA-binding proteins that modulate the flow of genetic information from DNA to RNA.1 TFs can recognize small degenerate DNA sequences (6−12 bp) within the gene regulatory region and generally function in combinatorial manners to regulate the transcription of target genes. 2 Regular transcription is indispensable for normal cellular function, accurate embryonic development,3,4 the maintenance of cell type- and tissuespecific gene expression,5−7 and the response to cellular signaling,8,9 whereas the transcription misregulation10 and the mutations in transcription factors11 might lead to a variety of diseases such as cancer,12 diabetes,13 congenital heart disease,14 autoimmune diseases,15,16 and developmental syndromes.2 Owing to the pivotal role of TFs in gene expression as well as its close relationship with human diseases, the accurate measurement of transcription factors is of great importance to both biological research and medical diagnosis. The conventional methods for transcription factor measurement are electrophoretic mobility shift assay (EMSA)17 and footprinting analysis.18 Despite their widespread applications, © 2014 American Chemical Society
these methods are generally tedious and cumbersome with low throughput.19,20 The enzyme-linked immunosorbent assay (ELISA) is relatively sensitive,21 but it requires the specific antibody against the target. To overcome these problems, a homogeneous molecule beacon assay has been developed.19 However, its application is limited due to its reliance on the rational design of a covalent break within the TF binding site.22,23 To improve the detection sensitivity, some DNA amplification-based approaches have been developed, including the real-time polymerase chain reaction (PCR) assay,24 the isothermally exponential amplification-based colorimetric assay,20 the target-converted helicase-dependent amplification assay,25 and the near-infrared fluorescent solid phase-based rolling circle amplification.26 These methods can amplify the TFs signal effectively. In view of the large numbers of transcription factors being identified27 and their nonuniform expression in different tissues,5,28,29 a versatile and sensitive Received: March 27, 2014 Accepted: May 27, 2014 Published: May 27, 2014 6006
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England Biolabs (Beverly, MA). The T7 RiboMAX Express Large Scale RNA Production System was obtained from Promega (Madison, WI,). Both SYBR Gold and SYBR Green I was purchased from Xiamen Bio-Vision Biotechnology (Xiamen, China). Protein−DNA Interaction and Exo III Digestion. The NF-κB p50 protein at various concentrations were mixed with 1 μL of 10 μM probes in 10 μL of binding buffer (10 mM TrisHCl, pH 7.5, 100 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mg/mL yeast tRNA, 10% glycerol, 0.25 mM DTT) and incubated at 37 °C for 20 min. Then 0.5 μL of Exo III (100 U/ μL) and 1 μL of 10 × NEB buffer 1 were added to the mixture and incubated for another 20 min at 37 °C to remove the free probes, followed by heating at 80 °C for 10 min to inactive Exo III. DNA Transcription. Before in vitro transcription, 1 μL of Exo I (20 U/μL) was added to the Exo III-treated solution and incubated at 37 °C for 20 min to remove the overhangs of the probes, followed by termination at 80 °C for 10 min. The DNA transcription was carried out in 20 μL of solution containing 2 μL of the Exo I-digested products at 37 °C for 60 min in T7 RiboMAX Express Large Scale RNA Production System (Promega, USA). EXPAR. The EXPAR reaction mixture was composed of part I and part II. Part I contained 2 μL of RNA transcription products, 2 μL of EXPAR template (1 μM), 0.5 μL of deoxynucleotide triphosphates (10 mM), and 2 μL of 10× NEB buffer 2. Part II contained 0.8 μL of Nt.BstNBI nicking endonuclease (10 U/μL) and 0.5 μL of Vent (exo−) DNA polymerase (2 U/μL) and 12.2 μL of H2O. The reaction was carried out in 20 μL of reaction solution. Before mixing with part II, part I was heated at 95 °C for 3 min. After the mixing of part I with part II, the reaction was performed at 55 °C for 50 min. The obtained products were kept at 4 °C for further use. Real-Time Fluorescence Measurement and Chemiluminescence Assay. The real-time fluorescence measurement was performed in the Roche Light Cycler Nano (Switzerland), and the fluorescence signal was recorded at a time interval of 30 s with SYBR Green I as the fluorescent indicator. For chemiluminescence assay, 20 μL of luminol solution (2.5 mM) and 40 μL of hemin solution (250 nM) were mixed with 20 μL of EXPAR products, 20 μL of distilled H2O, and 75 μL of reaction buffer (40 mM HEPES, pH 8.0, 300 mM NaCl, 20 mM KCl), followed by incubation at room temperature for 30 min to allow the obtained DNAzymes to fold into active quadruplex structure. Then 25 μL of H2O2 (80 mM) was added to the above solution, followed by the measurement of chemiluminescence signals using the GloMax 96 Microplate Luminometer (Promega, Madison, WI) with an interval of 1.5 s. Preparation of HeLa Cell Nuclear Extracts. HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, USA) with 10% fetal bovine serum (FBS; Invitrogen, USA) and 50 U/mL of penicillin plus 50 μg/ mL of streptomycin at 37 °C with 5% CO2. For the H2O2treatment experiment, the HeLa cells were treated with 500 μM H2O2 for 30 min before harvesting. The nuclear extracts were prepared using the nuclear extract kit (ActiveMotif, Carlsbad, CA), and the concentration of proteins was measured using Bradford-based assay. The activity of endogenous NF-κB p50 was determined by EMSA as well. Briefly, 2 μL of detecting probe (10 μM) and the nuclear extracts were incubated in 20 μL of binding buffer (10 mM Tris-HCl, pH 7.5, 100 mM KCl,
method that can measure transcription factors with a large dynamic range is highly desired. Recently, the functional G-quadruplex DNAzymes are widely used to measure a variety of targets owing to their high sensitivity, easy synthesis, and the capability to minimize nonspecific adsorption.30,31 Upon complexation with hemin,32 the G-quadruplex DNAzymes exhibit an enhanced horseradish peroxidase (HRP)-mimicking activity for 2,2′-azino-bis(3ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS) and luminol, generating a colored product32 and a chemiluminescence signal,33 respectively. The DNAzymes have been used to measure DNAs,30,33 metal ions,34,35 proteins,36 and the monitoring of telomerase activity.37 In this research, we demonstrate a label-free method for ultrasensitive measurement of transcription factors by integrating transcription-mediated dual isothermally exponential amplification reaction and Gquadruplex DNAzyme-driven chemiluminescence. Due to the cascade signal amplification of RNA polymerase-mediated transcription,38 the dual isothermally exponential amplification reaction,20,39 and the G-quadruplex/hemin DNAzyme-catalyzed chemiluminescence,33 the proposed method can detect NF-κB p50 with an extremely low detection limit of 6.03 × 10−15 M and a large dynamic range over 5 orders of magnitude, and it can be further applied to measure endogenous NF-κB p50 of crude cell nuclear extracts.
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EXPERIMENTAL SECTION Materials. The oligonucleotides (Table 1), RNase inhibitor, DEPC-treated water, and four mixed nucleotides (dNTPs) Table 1. Sequences of Synthesized Oligonucleotidesa note p50-Sb p50-ASc m-p50-S m-p50-AS EXPAR template synthesized DNAzyme
sequence (5′−3′) AGA TGG GAC TTT CCT TGC TAA TAC GAC TCA CTA TAG GGT CTC AGT CGT AGT GAG TCT GTT CTT GAT AAC AGA CTC ACT ACG ACT GAG ACC CTA TAG TGA GTC GTA TTA GCA AGG AAA GTC CCA TCT AGA TCT CAC TTT CCT TGC TAA TAC GAC TCA CTA TAG GGT CTC AGT CGT AGT GAG TCT GTT CTT GAT AAC AGA GTC ACT ACG ACT GAG ACC CTA TAG TGA GTC GTA TTA GCA AGG AAA GTG AGA TCT CCC AAC CCG CCC TAC CCA ACA GAC TCA CTA CGA CTG AGA CCC AAC AGA CTC ACT ACG ACT GAG ACC C−P GGG TAG GGC GGG TTG GG
a The boldface bases represent the binding region of NF-κB p50, and the underlined bold bases indicate the mutant bases. The italic bold bases indicate the T7 promote sequence. The underlined bases within the EXPAR template are the specific recognition sites of Nt.BstNBI nicking endonuclease. bThe letter S refers to the sense strand of the probe. cAS refers to the antisense strand of the probe.
were obtained from Takara Biotechnology Co. Ltd. (Dalian, China). To prepare the double-stranded detecting probe, two relevant single-stranded oligonucleotides were mixed at an equal molar ratio in the hybridization buffer (50 mM Tris− HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA). After heating at 95 °C for 5 min, the mixture was cooled to room temperature. The NF-κB p50 protein and c-Jun protein were purchased from Cayman Chemical (Ann Arbor, MI) and ProteinOne (Bethesda, MD), respectively. The exonuclease III (Exo III), exonuclease I (Exo I), Vent (exo−) DNA polymerase, and the Nt.BstNBI nicking endonuclease were purchased from New 6007
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endonuclease Nt.BstNBI,39 generating abundant DNA triggers and DNAzymes. Meanwhile, the released DNA triggers can further react with the EXPAR templates to initiate the next EXPAR. This dual EXPAR reaction will release large amounts of free DNAzymes to catalyze the H2O2-mediated oxidation of luminol, yielding an enhanced chemiluminescence signal in the presence of hemin. Conversely, in the absence of the TF, the probes will be degraded by exonuclease III and exonuclease I, resulting in neither the transcription-mediated EXPAR nor the DNAzyme-driven chemiluminescence signal. As a proof of concept, we demonstrated the quantitative measurement of transcription factor NF-κB p50. Validation of the Assay. The primary aspect of this assay is the conversion of the TF signal into RNA molecules. To determine whether the binding between TF and the probe can prevent the probe from Exo degradation and subsequently initiate the transcription reaction, we used a nondenaturing gel electrophoresis to monitor the transcription product generated by T7 RNA polymerase.38 As shown in Figure 1A, two distinct
2 mM MgCl2, 0.1 mM EDTA, 0.1 mg/mL yeast tRNA, 10% glycerol, and 0.25 mM DTT) at room temperature for 30 min. After labeling with SYBR Gold (1 μg/mL), the mixture was resolved on a prerun 8% nondenaturing polyacrylamide gel at 110 V in Tris-borate-EDTA (TBE) buffer (9 mM Tris-HCl, pH 7.9, 9 mM boric acid, and 0.2 mM EDTA). The gel was scanned by the Kodak 4000 MM imaging station (Kodak, Japan). Gel Electrophoresis. The products of two-stage isothermal amplification were resolved on a 12% nondenaturing polyacrylamide gel and run at room temperature in TBE buffer (9 mM Tris-HCl, pH 7.9, 9 mM boric acid, 0.2 mM EDTA) with a 110 V constant voltage for 50 min. The gel was stained by SYBR Gold and scanned with the Kodak Image Station 4000 MM.
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RESULTS AND DISCUSSION Principle of Transcription Factor Assay. The proposed method for transcription factor assay involves the TF-binding probe, exonuclease III/I (Exo III/I), T7 RNA polymerase, EXPAR template, and HRP-mimicking DNAzyme of PW17 (Scheme 1). The TF-binding probe is a double-stranded DNA
Scheme 1. Schematic Representation of Transcription Factor Assaya
a
This method combines the transcription-mediated dual exponential amplification reaction with the DNAzyme-catalyzed chemiluminescence.
Figure 1. (A) Analysis of the transcription products by nondenaturing PAGE. Lanes M, 1, and 2 show the DNA ladder marker, the products without the addition of NF-κB p50, and the products with the addition of 10 nM NF-κB p50, respectively. (B) Analysis of the EXPAR products by nondenaturing PAGE. Lanes M, 1, and 2 show the DNA ladder marker, the products without the addition of NF-κB p50, and the products with the addition of 1 nM NF-κB p50, respectively. (C) Time-dependent fluorescence signals obtained from dual EXPAR reaction in response to 1 nM NF-κB p50 (red line) and the control group without NF-κB p50 (black line). (D) Quantification of DNAzyme-catalyzed chemiluminiscence signal in response to 1 nM NF-κB p50 (red column) and the control group without NF-κB p50 (black column). Error bars are standard derivation obtained from three independent experiments.
in which the sense strand bears a specific TF recognition element and the T7 promoter sequence at the 5′ terminus and exonuclease III-resistant 3′-protruding termini (6 bases) at the 3′ terminus (p50-S in Table 1). In the presence of TFs, the binding of the probes with TFs prevents the probes from degradation by exonuclease III and exonuclease I (exonuclease III can remove the mononucleotides from 3′-hydroxyl termini of a duplex DNA,40 while exonuclease I can degrade the singlestranded DNA41,42). The probes with blunt ends can be efficiently transcribed by T7 RNA polymerase to yield abundant RNA,38 which is complementary to region I of the EXPAR template, and subsequently trigger the EXPAR reaction. The EXPAR template (Table 1) is a single-stranded DNA that contains three regions separated by two nicking endonuclease cutting sites, where regions I and II have an identical sequence and region III is complementary to the sequence of PW17 DNAzyme (see synthesized DNAzyme in Table 1). Upon hybridization with the EXPAR template, the transcribed RNA initiates the RNA-induced EXPAR with the assistance of Vent (exo−) DNA polymerase and nicking
bands of 25 nt and 60 bp, which represent the transcription product and the blunt probe, respectively, are observed with the addition of NF-κB p50 (Figure 1A, lane 2). However, neither the templates nor the RNA amplicons can be observed in the control group without the addition of NF-κB p50 (Figure 1A, lane 1). To verify whether the transcribed RNA amplicons can trigger the subsequent EXPAR reaction,39 we further measured the products of the EXPAR reaction with a nondenaturing PAGE. As shown in Figure 1B, a distinct band of G-quadruplex 6008
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helicase-dependent amplification method (0.93 pM).25 The improved sensitivity of the current assay might be ascribed to two factors: (1) the cascade signal amplification induced by DNA transcription,38 EXPAR reaction, and G-quadruplex DNAzyme-driven chemiluminescence33 and (2) abundant DNAzymes produced by the transcription-mediated dual EXPAR amplification.20,39 Specificity of the Assay. The specificity of current assay depends primarily on the specific binding of TF with the probes. To investigate the detection specificity, we measured the chemiluminescence intensity induced by a nonspecific binding probe, in which the p50 binding sequence of GGG AC TTT was replaced by CTC AC TTT (Table 1) and two distinct proteins including an irrelevant protein BSA20 and an oncogenic protein c-Jun45 under identical conditions (Figure 3). An extremely high chemiluminescence intensity is obtained
DNAzymes (17 nt) is observed with the addition of NF-κB p50 (Figure 1B, lane 2), while no distinguishable band of Gquadruplex DNAzymes is observed in the control group without the addition of NF-κB p50 (Figure 1B, lane 1), indicating that the transcribed RNA can initiate the EXPAR reaction effectively and specifically. These results are also confirmed by the real-time fluorescence assay (Figure 1C). The addition of NF-κB p50 induces an increasing fluorescence signal in the initial 50 min, followed by reaching a plateau (Figure 1C, red line). In contrast, no enhanced fluorescence is detected in the control group without the addition of NF-κB p50 (Figure 1C, black line). Furthermore, we examined whether the obtained DNAzymes can form an active Gquadruplex structure with the cofactor hemin to catalyze the luminol-H2O2 reaction (Figure 1D). A strong chemiluminescence signal is detected with the addition of NF-κB p50 (Figure 1D, red column), while no distinct chemiluminescence signal is detected in the control group without NF-κB p50 (Figure 1D, black column). Improved Sensitivity of the Assay. We further measured NF-κB p50 at different concentrations using the proposed chemiluminiscence assay. Under optimal conditions (Figure S1 in Supporting Information), the chemiluminescence (CL) signal increases with the concentration of NF-κB p50, and the CL intensity shows logarithmical correlation with the concentration of NF-κB p50 in the range from 10 fM to 1 nM (Figure 2). The correlation equation is I = 6.97 E6 + 4.84E5
Figure 3. Chemiluminescence signals induced by 1 nM NF-κB p50 and 1 μM nonspecific probes (control, black column), 1 nM BSA and 1 μM NF-κB p50-specific probes (BSA, blue column), 1 nM c-Jun protein and 1 μM NF-κB p50-specific probes (c-Jun, green column), and 1 nM NF-κB p50 and 1 μM NF-κB p50-specific probes (p50, red column), respectively. Error bars are standard derivation obtained from three independent experiments.
only in response to the coexistence of NF-κB p50 and NF-κB p50-specific probes (Figure 3, red column), whereas no significant chemiluminescence signal is detected in response to either a nonspecific probe (Figure 3, black column) or nonspecific proteins such as BSA (Figure 3, blue column) and c-Jun protein (Figure 3, green column), confirming the excellent specificity of the current assay toward NF-κB p50. Measurement of Endogenous NF-κB p50. The nuclear extracts contain various DNA-binding proteins that may interfere with the NF-κB p50 assay.24 We further challenged our method by measuring the endogenous NF-κB p50 in crude HeLa cell nuclear extracts, and we used the conventional EMSA assay to validate the activity of NF-κB p50 as well (Figure 4). Without the addition of NF-κB p50-specific probes, no distinguishable band is observed in crude nuclear extracts (Figure 4A, lane 1). While with the addition of NF-κB p50specific probes, two distinct bands of NF-κB p50-probe complexes and free probes are observed (Figure 4A, lane 2), and a remarkable chemiluminescence signal is obtained correspondingly (Figure 4B, black column), suggesting the feasibility of the current method for cellular NF-κB p50 assay. Interestingly, the treatment of HeLa cells with H2O2, which can induce oxidative stress in the cells, generates a stronger band of NF-κB p50-probe complex (Figure 4A, lane 3) and correspondingly a higher chemiluminescence signal (Figure
Figure 2. Chemiluminescence (CL) intensity shows a linear relationship with the logarithm of the concentrations of NF-κB p50. Error bars are standard derivation obtained from three independent experiments.
log10 C (R2 = 0.9945), where I and C represents the CL intensity and the concentration of NF-κB p50, respectively. The calculated detection limit is 6.03 × 10−15 M, which is 10-fold lower than that obtained by real-time fluorescence assay (Figure S2 in Supporting Information). This enhanced sensitivity may be ascribed to the HRP-mimicing property of the DNAzyme−hemin complex which can catalyze the H2O2luminol reaction to enlarge the signal.33 It is worth noting that the sensitivity of current assay has improved by 6 orders of magnitude in comparison with those of molecular beacon-based FRET assay (20 nM),19 the label-free luminescence switch-on method (30 nM),43 and the gold nanoparticle-based colorimetric assay (10 nM).44 In addition, the sensitivity of the current assay has improved by 2 orders of magnitude in comparison with the isothermally exponential amplificationbased colorimetric assay (3.8 pM)20 and the target-converted 6009
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ACKNOWLEDGMENTS We gratefully acknowledge the financial supports from the Natural Science Foundation of China (Grants 21325523), the Award for the Hundred Talent Program of the Chinese Academy of Sciences, and the Fund for Shenzhen Engineering Laboratory of Single-molecule Detection and Instrument Development (Grant No. (2012)433).
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Figure 4. (A) EMSA analysis of the activity of endogenous NF-κB p50 in HeLa cell nuclear extracts. Lane 1 is 10 μg of nuclear extracts without the detecting probes, lane 2 is 10 μg of nuclear extracts with 1 μM NF-κB p50-specific probes, and lane 3 is 10 μg of H2O2-treated nuclear extracts with 1 μM NF-κB p50-specific probes. (B) Measurement of the chemiluminescence signals in normal HeLa cell nuclear extracts (black column), and H2O2-treated HeLa cell nuclear extracts (red column). Error bars are standard derivation obtained from three independent experiments.
4B, red column) than the untreated cells (Figure 4A, lane 2 and Figure 4B, black column), suggesting the enhanced activity of endogenous NF-κB p50 induced by oxidative stress46 as well as the feasibility of the current method for the measurement of NF-κB p50 in real samples.
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CONCLUSIONS In conclusion, we have demonstrated a label-free and ultrasensitive method for transcription factor assay using transcription-mediated dual EXPAR and G-quadruplex DNAzyme-driven chemiluminescence. The specific binding of TF with the detecting probes can not only ensure the detection specificity, but also trigger the subsequent cascade signal amplification of RNA transcription, dual EXPAR, and the DNAzyme-catalyzed chemiluminescence. The detection limit of current assay can reach as low as 6.03 × 10−15 M, which is more sensitive than those of previously reported methods.19,20,25,43,44 Notably, the detecting probe used in this research does not require any modification, and the entire reaction can be carried out in an isothermal manner without the involvement of highprecision thermal cycler, making current assay extremely costeffective. More importantly, this method can be used to measure a variety of DNA-binding proteins by simply substituting the target-specific DNA-binding sequence in the detecting probes.
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ASSOCIATED CONTENT
S Supporting Information *
Optimization of experimental conditions and the real-time fluorescence measurement. This material is available free of charge via the Internet at http://pubs.acs.org.
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REFERENCES
AUTHOR INFORMATION
Corresponding Author
*Phone: +86-755-8639-2211. Fax: +86-755-8639-2299. E-mail: [email protected]. Author Contributions †
These authors contributed equally.
Notes
The authors declare no competing financial interest. 6010
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